The long-term objective of this research is to develop physical maps of human chromosomes beginning with regions around the loci for particular inherited diseases. The specific aims of the proposed work are to build a comprehensive library of yeast clones carrying human DNA on linear artificial chromosomes, to isolate overlapping clone carrying DNA spanning the loci for four inherited human diseases, and to identify the gene for one of those diseases. The availability of cloned human DNA spanning these loci will permit the development of reliable diagnostic tests for inheritance of these diseases based on the use of tightly linked polymorphic DNA probes. Identification of the gene for an inherited disease offers an opportunity to understand the molecular defect involved and eventually to develop more effective treatments. In the long term, information on organization of restriction fragment length polymorphisms and restriction sites in the human genome derived from this work will be helpful for building a physical map and subsequently sequencing the genome. In the course of this work, the yeast artificial chromosome cloning methods will be modified to increase the frequency of obtaining clones with large DNA inserts, to permit isolation of clones which overlap on either side, to prevent polymerization of insert DNA, and to permit purification of the artificial chromosome. DNA probes genetically linked to the genes for neurofibromatosis 1, adult polycystic kidney disease familial polyposis, and multiple endocrine neoplasia (type 2A) will be used to isolate yeast clones from a library representative of the whole human genome. The actual disease locus will be approached from these clones by using new "walking" and "locus expansion" techniques. The position of clones relative to the disease loci will be determined by development of polymorphic DNA markers and measurement of linkage with the disease gene segregating in affected families. Identification of the gene for familial polyposis will be based on screening for detectable alterations in the cDNA or the gene in tissues from affected individuals.